Recombinant modified bacillus anthracis protective antigen for use in vaccines
a technology of recombinant modified bacillus anthracis and protective antigen, which is applied in the direction of antibody medical ingredients, immunological disorders, peptide sources, etc., can solve the problems of no defined vaccine-induced protective level and difficult standardization
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example 1
[0105]In this example, the expression and the stability of two recombinant PA variants, PA-SNKE-ΔFF-E308D (SEQ ID NO: 4) and PA-N657A (SEQ ID NO: 5), were studied. These proteins were expressed in the non-sporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-ΔFF-E308D (SEQ ID NO: 4), which lacks two proteolysis-sensitive sites, is more stable than PA-N657A (SEQ ID NO: 5). Process development was conducted to establish an efficient production and purification process for PA-SNKE-ΔFF-E308D (SEQ ID NO: 4). Various parameters such as pH, media composition, growth strategy, and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only sources of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was found to be 14-18 hours after inoculation, and EDTA (5 mM) was added upon harvesting for proteolysis control. In one of the processes described herein, ...
example 2
Composition of the Vaccines
[0139]Four combinations of the recombinant (modified) protective antigen (“rPA”) were made: (1) rPA in PBS (“phosphate buffered saline”), (2) rPA in formalin, (3) rPA in aluminum hydroxide and (4) rPA in formalin and aluminum hydroxide. Another formulation of succinylated rPA was prepared and tested (data not shown).
example 3
Immunogenicity in Mice
[0140]The four formulations described above were immunogenic in mice, and induced antibody levels comparable to those induced by the currently licensed anthrax vaccine. The induced antibodies had anthrax toxin neutralizing activity. It is planned to evaluate these formulations in humans, and to choose the best one for use as a vaccine.
[0141]The data from the mice experiments are set forth in the tables 3 to 5 below:
TABLE 3Number of Mice and ImmunogenGroup NumberNumber of MiceImmunogen105611PA (2.5 μg)-Untreated105711PA (12.5 μg)-Untreated105811PA (2.5 μg) + Alum105910PASUCC 10:1.25 (2.5 μg)106010PASUCC 10:1.25 (12.5 μg)106110PASUCC 10:3 (2.5 μg)106210PASUCC 10:3 (12.5 μg)106310PA-Formalin 0.3 (2.5 μg)106410PA-Formalin 0.3 (12.5 μg)106510PA-Formalin 3.0 (2.5 μg)106610PA-Formalin 3.0 (12.5 μg)106710PA-Formalin 7.12 (2.5 μg)106810PA-Formalin 7.12 (12.5 μg)106911Anthrax Vaccine 0.1 ml107010Control
TABLE 4Antibody Levels and Neutralization TitersMiceμg / mlNeutral, Tit...
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